In 2010, we have advanced the objectives of this project as follows: (1) Rotavirus genotype G1:P8 is the most commonly detected G-P combination globally. In addition, a monovalent vaccine containing genotype G1:P8 virus has been licensed in many countries worldwide. Increasing evidence suggests that genetic drift observed among community G1 strains is generating antigenic variants, an observation which may be important for the development of vaccination strategies. The human G1 rotavirus VP7 gene, which encodes the major protective and neutralization antigen, has been reported to belong to one of at least four phylogenetic lineages. The recently licensed monovalent vaccine contains a lineage 2 G1 virus whereas another licensed pentavalent reassortant vaccine contains a lineage 3 G1 virus. To study the relationship between the phylogenetic lineages and the neutralization specificity of selected G1 strains belonging to lineage 1, 2, 3 or 4, we generated (i) single gene substitution reassortants each of which bore 10 genes including the VP7 gene of G1 rotavirus strain and only the VP4 gene of bovine UK strain;or 10 genes of bovine UK strain and only the VP7 gene of G1 rotavirus strain belonging to one of four VP7 gene lineages (2 strains each of lineage 1, 2 or 3;and 3 lineage 4 strains);and (ii) hyperimmune guinea pig antiserum to each reassortant. Preliminary data obtained by two-way cross-neutralization assays suggest that, unlike G9 rotavirus VP7s which display lineage-specific neutralization profiles, no significant difference in neutralization specificities is detected among the four G1 VP7 lineages tested. (2) In a study performed in 1983, 18 adult volunteers received oral challenge with the virulent human rotavirus strain D (G1P1A.8,NSP4B). To identify correlates of resistance to rotavirus infection, we analyzed levels of serum immunoglobulin (Ig) A and IgG antibodies to various rotaviral antigens in 16 of the 18 volunteers. We used immunocytochemical assays that involved a total of 16 different recombinant baculoviruses, with each baculovirus expressing one of the following major serotype/genotype rotavirus proteins for the serologic assays: (i) viral protein (VP) 4 with P1A.8, P1B.4, P2A.6, P3.9, or P4.10 specificity;(ii) VP7 with G1-G4 or G9 specificity;and (iii) nonstructural viral protein (NSP) 4 with genotype A, B, C, or D specificity. We demonstrated that the prechallenge titers of IgG antibody to VP7 types G1, G3, G4, and G9;VP4 types P1A.8,P1B.4,P2A.6, and P4.10;and NSP4 type A in the group of noninfected volunteers (n=11) were significantly higher than those in the group of infected volunteers (n=5;of these 5 volunteers, 4 were symptomatically infected). Moreover, logistic regression analysis showed that resistance to rotavirus infection most closely correlated with higher prechallenge titers of IgG antibody to homotypic VP7 (G1) and VP4 (P1A.8). These results suggest that protection against rotavirus infection and disease is primarily VP7/VP4 homotypic and, to a lesser degree, heterotypic. (3) Using an immunocytochemical staining assay involving six different recombinant baculoviruses with each expressing one of the major bovine rotavirus VP7 (G6, G8 and G10) and VP4 (P6.1, P7.5 and P8.11) serotypes, we analyzed IgG antibody responses to individual proteins in archival serum samples collected from 31 calves monthly from 1 to 12 months of age during 1974-1975 in Higley, Arizona. Seroresponses to VP7 and VP4, as determined by a fourfold or greater antibody response, were not always elicited concurrently following infection: in some calves, (i) seroresponses to VP7 were detected earlier than to VP4 or vice versa;and (ii) a subsequent second seroresponse was detected for VP7 or VP4 only. In addition, a second infection was more likely to be caused by different G and/or P types. Analyses of serum samples showed that the most frequent GP combination was G8P6.1, followed by G8P7.5, G8P8.11 and G6P6.1. (4) It is universally acknowledged that genome segment 4 of group A rotavirus, the major etiologic agent of severe diarrhea in infants and neonatal farm animals, encodes outer capsid neutralization and protective antigen VP4. To determine which genome segment of three group A equine rotavirus strains (H-2, FI-14 and FI-23) with P12 specificity encodes the VP4, we analyzed dsRNAs of strains H-2, FI-14 and FI-23 as well as their reassortants by polyacrylamide gel electrophoresis (PAGE) at varying concentrations of acrylamide. The relative position of the VP4 gene of the three equine P12 strains varied (either genome segment 3 or 4) depending upon the concentration of acrylamide. The VP4 gene bearing P3, P4, P6, P7, P8 or P18 specificity did not exhibit this phenomenon when the PAGE running conditions were varied. Thus, we demonstrated that the concentration of acrylamide in a PAGE gel affected the VP4 gene coding assignment of equine rotavirus strains bearing P12 specificity.